title: Dual ligation hybridization assay for the specific determination of oligonucleotide therapeutics.
abstract: BACKGROUND
  Oligonucleotide-based therapeutics are quantified with hybridization assays in biological matrices such as plasma and tissues. Current hybridization methods do not entirely discriminate the parent compound from 5´- or 3´-N-X truncated metabolites.
  
  
  RESULTS
  A dual ligation-based hybridization assay was developed to circumvent the limitations of current assay formats. Ligation of probes at either end of the analyte is performed via a bi-enzymatic reaction consisting of polynucleotide kinase and DNA ligase. The method was validated with regard to mechanism, specificity, precision and accuracy.
  
  
  CONCLUSIONS
  The dual ligation assay is specific for the parent compound and detects the full-length product with intact 5´- and 3´-ends. The dual ligation assay can also be used to specifically determine individual metabolites in complex mixtures and is currently implemented to quantitative PCR.
authors.affiliation: Charles River Preclinical & Clincal Services Montréal Inc., 22022 Transcanadienne, Senneville, QC H9X3R3, Canada. guy.tremblay@crl.com
authors.forename: Ga
authors.initials: G
authors.lastname: Tremblay
authors.forename: G
authors.initials: G
authors.lastname: Khalafaghian
authors.forename: J
authors.initials: J
authors.lastname: Legault
authors.forename: P
authors.initials: P
authors.lastname: Nielsen
authors.forename: Aj
authors.initials: A
authors.lastname: Bartlett
mesh_terms: D001483:Base Sequence; D002138:Calibration; D011088:DNA Ligases / Q000378:metabolism; D006801:Humans; D007118:Immunoassay; D008024:Ligands; D016014:Linear Models; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000627:therapeutic use*; D016133:Polymerase Chain Reaction; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D008024:Ligands; D009841:Oligonucleotides; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D011088:DNA Ligases